The organization of gonadal primordia into testes or ovaries is the first event of sexual differentiation. In mammals, gene(s) on the Y chromosome regulates testicular development, and in its absence, gonadal primordia develop into ovaries. It is the hormones produced by testes or ovaries that control the development of phenotypic sex. Thus, genetic sex coincides with gonadal sex and phenotypic sex when an individual undergoes normal development. However, patients have been described with gonads that do not coincide with their karyotyped sex. The objective of our study is to elucidate the regulation of gonadal differentiation in mammals, using the mouse as a model. The first specific aim is to identify the factors that induce testicular differentiation in ovarian primoridia. We found that fetal mouse ovaries develop testicular structures (ovotestes) when transplanted under the kidney capsules of adult male mice, but not when transplanted under the kidney capsules of adult female mice. The testicular structures of ovotestes are comparable to those of normal testes. We will determine whether ovostestes have testicular functions such as production of testosterone and expression of Beta-endorphin, a biochemical marker for Leydig cells. We will assess the relation of histocompatibility-Y (H-Y) or serologically detectable male (SDM) antigen to testicular differentiation by transplanting fetal ovaries into mutant female mice which have ovaries and are typed as H-Y or SDM antigen positive. We will also determine the role of germ cells in ovotestis development by transplanting germ-cell-deficient ovaries into male mice. Our goals are to establish and in vitro system in which ovotestes develop, and to clarify how ovarian primordial cells differentiate into testicular components. The second specific aim is to determine the essential nutrients for testicular organization in vitro. We found that gonadal primordia differentiate into testes or ovaries in vitro only in the presence of serum. We will characterize the active components of human serum required for organization of testis cords and survival of germ cells, respectively. From these studies, we expect to acquire a better understanding of the regulation of gonadal differentiation in mammals, as well as the disorders of sex development in humans.